Dictyostelium discoideum is a primitive eukaryotic microorganism commonly called a slime mold, or more specifically, a cellular slime mold. The name is derived from the two extreme states of the microorganism from a macroscopic perspective. When actively growing, D. discoideum grows as single cell amoeba. At this stage the organism has no cell wall, hence it appears as a thin film (or slime). Upon starvation on a solid medium, the independent cells aggregate to form a colony. The colony exhibits traits of a multicellular organism in that it migrates in the form called a slug and then differentiates, with the posterior cells of the slug forming a foot, the anterior cells forming a stalk and the middle cells forming a fruiting body. The organism is found naturally on the surface of soil and dung. The wild type amoeba obtains nutrients exclusively by ingestion (phagocytosis) of whole bacteria; for this reason, the organism is sometimes referred to as carnivorous. Axenic mutants of D. discoideum have been isolated which are capable of growth without coculture of "food" bacteria and which therefore can be grown on soluble media. The present invention relates to the immobilization and use of a novel dipeptidylaminopeptidase (DAP) isolated from D. discoideum.
Dipeptidylaminopeptidases are enzymes which hydrolyze the penultimate amino terminal peptide bond releasing dipeptides from the unblocked amino-termini of peptides and proteins. There are currently four classes of dipeptidylaminopeptidases (designated DAP-I, DAP-II, DAP-III and DAP-IV) that differ based on their physical characteristics and the rates at which they react with their substrates. DAP I is a relatively non-specific DAP that catalyzes the release of many dipeptide combinations from the unblocked amino termini of peptides and proteins. DAP I shows little or no activity if the emergent dipeptide is X-Pro, Arg-X, or Lys-X (where X is any amino acid). DAP II shows a preference for amino terminal dipeptide sequences that begin with Arg-X or Lys-X, and to a lesser extent, X-Pro. DAP-II exhibits significantly lower reaction rates versus most other dipeptide combinations. DAP III appears to have a propensity toward amino terminal dipeptide sequences of the form Arg-Arg and Lys-Lys. DAP IV shows its highest rate of hydrolyric activity toward dipeptide sequences of the form X-Pro. The DAP enzymes, particularly DAP-I and DAP-IV, have been shown to be useful in processing proteins.
The present invention concerns a novel DAP isolated from Dictyostelium discoideum (dDAP), which is the subject of U.S. patent application Ser. No. 07/955,539. More significantly, the present invention represents an improved method for processing proteins having an even numbered amino acid N-terminal extension. The older method is a single-use batch reaction in which dDAP is added to substrate and allowed to react for a specified time. Subsequent purification steps were needed to ensure removal of dDAP from the product stream. The improvement over single-use batch conversion reactions is a result of immobilizing dDAP to relatively inexpensive and commercially available chromatography resins to selectively immobilize dDAP in an active form.
It was most surprisingly found that dDAP strongly binds to anion exchange resins under acidic conditions where most proteins either do not bind or only bind weakly. Under acidic conditions where anion exchange resins typically are not used, dDAP is both stable and highly active against certain substrates of interest. The acidic pH effectively prevents the subsequent binding of substrate and product to this resin at levels high enough to impact yield or at levels high enough to displace the non-covalently attached enzyme. The effective concentration of dDAP on the resin can be made quite high enabling relatively rapid reaction kinetics. The present invention decreases the time that reactants and products are exposed to harsh conditions of pH and temperature that adversely impact reactant and product stability, yield, purity, and structure. Moreover, the present process provides a convenient means for reusing dDAP in later conversion reactions. Thus, the benefit of the improved process of the present invention is to reduce the overall amount of enzyme required to convert a given amount of precursor protein and to reduce the time that reactants and products are exposed to harsher batch process conditions.